DNA sequencing continues to be an important analytical tool for providing information relevant to laboratory research, biotechnology, clinical medicine, and drug discovery. In one method, the Maxam-Gilbert chemical degradation method, the DNA molecule to be sequenced is labeled at one end with a radioisotope, and then apportioned to four different reactions which together, are specific for all four bases. In each reaction, the DNA is cleaved at a specific nucleotide base (either guanine (G), cytosine (C), thymine (T), or adenine (A)). The four different sets of fragments are then electrophoresed to separate the fragments into bands based on their length, thus identifying the 3'-terminii positions defined by the cleavage of the given nucleotide base. In another method, the Sanger dideoxy chain termination method, the DNA sequence of a single-stranded template DNA is determined by using the template to synthesize multiple copies of the complementary strand. A primer is used to anneal to the template and to provide an initiation point from which synthesis proceeds. Typically, the annealed primer-template is added to four reactions, each of which is combined with four deoxynucleotide triphosphates ("dNTPs", e.g., dATP, dTTP, dCTP, dGTP), a DNA polymerase enzyme, and a single dideoxynucleotide triphosphate ("ddNTP"). The reaction conditions are designed so that the ddNTP competes with the dNTPs for incorporation into the synthesized strand; that the incorporation of the ddNTP into the strand prevents further extension; that different size strands are synthesized; that the synthesized strands may be separated into discrete band by electrophoresis; and that the sequence can be determined from the pattern of bands because each band corresponds colinearly to a specific nucleotide.
Typically, radioisotopes have been, and continue to be, used to label nucleotides to visualize the bands resulting from electrophoresis. However, there are several disadvantages inherent to using a radioisotope to label the nucleotides for a sequencing reaction, including problems associated with storage, use, and disposal of radioisotopes. Handling and disposing of radioactive gels and radioactive wastes are problematic as to logistics, and also involves issues related to health and safety. Use of fluorescent labels has become more commonplace in sequencing reactions. Many current automated DNA sequencing methods utilize fluorescence detection of labeled synthesized DNA strands. One method of fluorescence labeling is to use a single fluorophore in the four sets of reactions to generate the synthesized strands which are then loaded into adjacent lanes and separated by electrophoresis. Use of a single fluorophore is disadvantageous because (a) automated scanning of four lanes can be difficult due to lane-to-lane variations in the migration velocity of electrophoresing synthesized strands; and (b) four lanes are needed, as each of the four reactions need be loaded into a separate lane, thereby reducing throughput.
There exists fluorescence-based DNA sequencing systems using fluorophores. U.S. Pat. No. 5,723,298 describes a method using two cycles of primer extensions, wherein the primer extension product is labeled by incorporating a fluorescent fluorophor-labeled dNTP. This technology is representative of the single-label/four-lane system. U.S. Pat. No. 5,436,130 describes the use of two different fluorescent dyes to label nucleotides used as terminators in strand synthesis. A system utilizing multi-color detection and different fluorescent dyes would be desirable. Such systems have been described previously, and have encountered various problems in application. For example, in one system the primers are labeled with four different dyes (Smith et al., 1985, Nucleic Acids Res. 13:23999-2412). However, use of the four dyes utilized appears to cause significant perturbations in electrophoretic mobilities, thereby making sequence interpretations difficult. Properties that may effect such perturbations include the difference in molecular size, charge, and shape of the dyes utilized.
Other systems have been proposed, wherein up to four different fluorescent dyes are used in a sequencing method, and wherein each dye has a different excitation wavelength and a different emission wavelength. However, one would expect to have to use more than one excitation light source to provide the necessary excitation wavelengths required (see, e.g., U.S. Pat. Nos. 5,675,155, and 5,650,277). Additionally, a large wavelength range (e.g., 100 nm between different excitation wavelength spectrum) for excitation of a combination of fluorescent dyes used in the same system makes it difficult to excite the combination with a single monochromatic light source. Further, a large dynamic range in the detectable signal intensities emitted by four different fluorescent dyes presents difficulties in the detection process. However, use of fluorescent dyes having closely spaced absorption (excitation) and corresponding emission wavelengths could present difficulties in distinguishing between the different emission spectra in order to identify the individual labeled synthesized strands.
Thus, there continues to be a need for a class of fluorescent molecules which can be used in combinations in preparing labeled nucleobases for nucleic acid molecule strand synthesis, and particularly for automated DNA sequencing. For example, in nucleic acid molecule synthesis and amplification, there remains a need for a nonisotopic detection system which can be used in the detection of nucleic acid molecules; and which utilizes a class of molecules that may be excited with a single excitation light source resulting in detectable fluorescence emissions of high quantum yield and with spectrally resolvable fluorescence peaks, thereby allowing simultaneous detection of several colors by an appropriate detection system.